Analyte detection system

ABSTRACT

There is disclosed an analyte detection system comprising: and analyte analysis stage through which a condensed phase containing the analyte flows; nebuliser means into which the output of the analyte analysis stage is introduced, the nebuliser means producing an analyte containing aerosol; a flame based analyte detector, and means for introducing the analyte containing aerosol into said flame based anylte detector; in which the condensed phase produces no or negligible response from flame based analyte detector, and I) the system can operate at condensed phase flow rates of greater than 20 μl min −1 , preferably greater than 50 μl min −1 , most preferably greater than 100 μl min −1 ; and/or ii) the means for introducing the analyte containing aerosol into said flame based detector comprises a spray chamber

This invention relates to an analyte detection system, in particular toa “universal” detection system in which an analyte analysis stage, suchas a liquid chromatography stage, is interfaced to a flame baseddetector, such as a flame ionisation detector (FID).

Traditionally, the direct detection of compounds (analytes) dissolved inflowing liquid streams, such as the eluent from liquid chromatography,or the effluent from flow injection analyses, has generally depended onspectroscopic methods, usually ultraviolet or fluorescence spectroscopy.These widely used approaches require the presence in the analyte of achromophore or fluorophore. However, many compounds of interest produceonly a low response or a negligible response using such detectionmethods. In addition many compounds which provide a signal can generatevery different mass responses (i.e., large differences in the magnitudeof the output signal for the same amount of analyte, because ofdifferences in extinction coefficients) so that direct individualcalibration of the detector is required.

A wider response can be obtained by using a mass spectrometry as adetector, but currently liquid chromatography-mass spectrometry linkedsystems (LC-MS) are rather expensive and would normally not be employedfor routine analyses. Similarly, the sample can be passed to a plasmatorch (an inductively coupled plasma-ICP), where an element specificresponse can be obtained by emission (ICP-Emission) or mass spectrometry(ICP-MS), but again this is a relatively expensive method and isprimarily applicable to analytes containing metal atoms rather thanorganic compounds. In each case the sample has to be evaporated, sprayedor nebulised into the gas phase so that it can be introduced into thedetection cell or plasma.

Ever since liquid chromatographic methods have been employed, there hasbeen a need for a so-called “universal detector”, which could provide aresponse which ignored the eluent mobile phase but detected thedissolved analytes and gave a similar signal magnitude for similarquantities of each analytes in a mixture.

Previously reported broadly applicable or universal detection methodshave primarily been based on the measurement of changes in therefractive index (RI) of the eluent, which tends to be a relativelyinsensitive method, because it depends on the measurement of smallchanges in the bulk properties of the eluent. The alternative approachhas been the light scattering detector (LSD) (or mass evaporativedetector), in which the eluent is sprayed into a chamber using an inertgas and the solvent is evaporated from the droplets leaving particles ofinvolatile analytes. These pass through a light beam from a lamp orlaser and the scattered light is detected. This detector has somelimitations, since the response can be non-linear as it depends onparticle size. This detector has the disadvantage that volatilecomponents of the analyte mixture are usually lost together with thesolvent so that only higher molecular weight analytes or involatilecomponents can be detected.

The primary alternative group of methods have employed variants of the“transport” detector, which involve a step in which eluent is lost orhidden during the transport of the analyte from the end of theseparation system to the detection step. The effluent is deposited oneither a moving chain, moving belt, moving wire, rotating disc orhelical wire and the solvent is evaporated (usually thermally and/orunder a reduced pressure), leaving the analyte to be transported to thedetection step. The detector in these cases is usually a flameionisation detector (FID) or mass spectrometer (MS). Both of thesedetectors provide a broadly universal response for those organicanalytes which reach the detector. Unfortunately, problems with thetransport detectors, such as inhomogeneous sampling, loss of volatileanalytes during solvent evaporation, spiking due to sample diffusion onthe hot metal conveyors causing an increase in background noise andghost peaks due to incomplete removal of analyte from reusable conveyorsystems have limited their application. The mass spectrometer and flameionisation detectors both provide a high energy to the analytes breakingthem down to form ions, which can be detected as a current flowingacross a potential difference. The primary difference is in thecomplexity of the system, the amount of information determined and hencethe cost.

The linkage of liquid chromatography (LC) to a FID (a detector commonlyassociated with gas chromatography) is highly attractive, as thedetector is much simpler to manufacture and operate than a massspectrometer. Also, the FID has a high sensitivity, is capable ofdetecting both volatile and non-volatile species, and does not require achromophore. The detector response is highly linear over a wide range ofsample masses and most organic compounds will have a similar massresponse, although compounds which do not burn are generally notdetected. However, the application of FID to LC has been limited becausemost separations employ mixtures of an organic solvent (or solvents) andwater as the eluent and the former causes a high background signal inthe detector as it can burn. Hence, transport detectors of the typediscussed above have been employed to remove the mobile phase before thedetection step. The FID is well known as the most successful universaldetector in gas chromatography where the inorganic carrier gas producesno response, but gas chromatography is limited to volatile and thermallystable analytes.

In the last few years, superheated (subcritical) water has been employedas the sole liquid component of the mobile phase for liquidchromatography, leading to a number of attempts to directly link thechromatographic separation stage to a flame ionisation detector.Superheated water is produced when water is heated at temperatures above100° C. under sufficient pressure to remain as a liquid. Under suchconditions, it can mimic the elution characteristics of organic solventsand organic/aqueous mixtures. Furthermore, superheated water produces anegligible background response from the FID.

Published work has employed two main approaches. The first method iseffectively based on the well known thermospray concept often used inearly LC-MS coupling (Miller, D. J. and Hawthorne, S. B., Anal. Chem.,69 (1997) 623; Smith, R. M.; Burgess, R J.; Chienthavom, O.; Stuttard,J. R., LC-GC International, 12 (1999) 30; Ingelse, B. A.; Janssen, H.G.; Cramers, C. A., HRC-Journal of High Resolution Chromatography, 21(1998) 613). These approaches employed a heated capillary tube, usuallymetal or glass, placed within the flame jet of the GC or very close toit. Generally a high temperature of about 300-400° C. is used causingthe eluting liquid to flash-evaporate, thereby spraying any volatilecomponents into the flame of the FID, where organic analytes burngenerating a conventional signal. Additionally, the capillary oftenprovides a back-pressure to the chromatographic system. A slightlydifferent approach has been to use a commercially available eluent jetinterface (Hooijschuur, E. W. J.; Kientz, C. E.; Brinkman, U. A., Th. J.High Res. Chrom., 23 (2000) 309). In this design the capillary containsa restriction close to the tip, which is inductively heated using80-90W, thus producing a sharp temperature gradient.

Both these concepts are based on heating the aqueous solution togenerate steam to transport the analyte into the gas phase and hence tothe detection flame. A disadvantage with these systems is that if theanalyte is involatile or thermally unstable, it can be deposited ordegraded and may rapidly block the capillary, thus reducing sprayefficiency. This appears to limit the useful response to analytes whichare thermally stable and volatile. The main problem associated withthese interfaces are the lack of reliability and robustness of theinstrumentation.

Hooijschuur et al, ibid, also describes a comparison experiment in whichan interface based on a microjet nebuliser is employed. In fact, it isthis comparison experiment which, with hindsight, bears the closestsimilarity to the present invention. However, Hooijischuur et al isquite negative in its assessment of this nebuliser based technique, andcompares the nebuliser based technique unfavourably with the eluentevaporation technique, which is the main thrust of Hooijschuur et al.

Furthermore, this comparison experiment is not a practical routinesystem, inter alia because a micro capillary liquid chromatography (LC)system is employed, together with a micro capillary linking the LCsystem to the nebuliser, thus limiting flow rates and hence sensitivity.Flow rates of 10 μl min⁻¹ are reported. A further problem is that, evenif higher flow rates were contemplated, condensed liquid would drain tothe bottom of the detector and be blown through the flame causingconsiderable noise. A third problem is that if larger aerosol dropletsare formed, they can cause spiking in the flame, since such largerdroplets are not prevented from travelling from the nebuliser to theflame. A fourth problem is the rather cumbersome arrangement of thecomparison experiment in which, apparently, the FID is positionedinverted below the nebuliser. It must be emphasised that this experimentwas used for comparison purposes with the heated capillary techniquewhich is the principal concern of Hooijschuur et al., and the comparisonwas unfavourable towards the nebulisation technique. Thus the prior artin which the use of superheated water is combined with FID detectionclearly points the skilled person towards flash evaporation of theeluent.

Further prior art is discussed below. GB 1475432 discloses an interfacefor liquid chromatography in which an oscillator is used to atomise theeluent from a chromatographic column. U.S. Pat. No. 5,153,673 disclosesa pulsed flame detector for use with numerous samples. U.S. Pat. No.3,967,931 discloses a system in which the eluent from a liquidchromatography column is aspirated directly into a flame, therebyproducing an aerosol in situ within the flame. A problem associated withthis approach is that non-volatile analytes can be degraded, causingdepositions and possible blockages.

The present invention overcomes the aforesaid problems anddisadvantages, and provides a practical, cost-effective “universal”detector.

According to a first aspect of the invention there is provided ananalyte detection system comprising:

-   -   an analyte analysis stage through which a condensed phase        containing the analyte flows;    -   nebuliser means into which the output of the analyte analysis        stage is introduced, the nebuliser means producing an analyte        containing aerosol;    -   a flame based analyte detector; and    -   means for introducing the analyte containing aerosol into said        flame based analyte detector;    -   in which: the condensed phase produces no or negligible response        from flame based analyte detector; and    -   i) the system can operate at condensed phase flow rates of        greater than 20 μl min⁻¹, preferably greater than 50 μl min,        most preferably greater than 100 μl min⁻¹;    -   and/or ii) the means for introducing the analyte containing        aerosol into said flame based detector comprises a spray        chamber.

By providing systems having this combination of features, it is possibleto produce a so-called “universal” detector which can be used to detectorganic and inorganic compounds, irrespective of whether the compoundsare volatile or non-volatile. Additionally, elements can be detected.Furthermore, the system is practical and cost effective. Furtherstill,the provision of nebulisation means as a way of producing an aerosolremoves the problems of deposition, decomposition and blockageassociated with high temperature evaporative methods, since nebulisationtakes place at a temperature substantially below the boiling point ofthe condensed phase.

The condensed phase may be aqueous, and may be an aqueous solution. In apreferred embodiment, the condensed phase is a super heated aqueoussolution. It is possible to include other components such as inorganicbuffers, ion-pair reagents, acids, bases and organic additives such asformic acid or trifluoroacetic acid provided that such components giverise to no or negligible signal from the detector. Other possiblesolvents are carbon tetrachloride, Freons such as 124a, and subcriticalcarbon dioxide.

The condensed phase may be a supercritical fluid, such as carbondioxide, xenon, argon, ammonia and nitrous oxide.

The condensed phase may be a superheated liquid phase, such as carbondioxide or, as mentioned above, water.

The detector may be a FID. This has the advantage of providing a linearresponse over a wide analyte mass range, and is relatively inexpensive.

The detector may be a thermionic detector, pulsed flame photometricdetector or flame photometric detector.

The analyte analysis stage may comprise an analyte separation stage, inwhich instance effluent from the analyte separation stage is introducedinto the nebulisation means.

The analyte separation stage may comprise a liquid chromatographicstage.

The analyte separation stage may comprise a capillary electrophoresis,electrochromatographic or capillary electrochromatographic stage.

Alternatively, the analyte stage many comprise a flow injection analysisstage.

The system may comprise a spray chamber in which separation of theaerosol is achieved so that larger aerosol droplets are not introducedto the flame based analyte detector. The spray chamber may be adapted tocreate turbulence in the flow of the aerosol. By removing largerdroplets, noise from the detector is reduced.

The spray chamber may be adapted so as to cause the aerosol to flow in acentrifugal flow pattern.

According to a second aspect of the invention there is provided the useof a system as according to the first aspect of the invention to detectvolatile and non-volatile compounds or elements.

According to a third aspect of the invention there is provided the useof a system according to the first aspect of the invention to detectorganic compounds.

According to a fourth aspect of the invention there is provided the useof a system according to the first aspect of the invention to detectinorganic compounds.

Systems and uses in accordance with the invention will now be describedwith reference to the accompanying drawings, in which:

FIG. 1 is a front view of an embodiment of a liquid chromatographysystem in accordance with the invention;

FIG. 2 shows a) front and b) side views of the spray chamber of FIG. 1;

FIG. 3 is a chromatogram showing carbohydrate separation;

FIG. 4 is a chromatogram showing amino acid separation; and

FIG. 5 is a chromatogram showing benzaldehyde detection usingsuperheated water as an eluent.

FIG. 1 shows an analyte detection system comprising:

-   -   a separation stage (shown generally at 10) through which a        condensed phase containing the analyte flows;    -   nebuliser means 12 into which the flowing condensed phase is        introduced, the nebuliser means 12 producing an analyte        containing aerosol;    -   a flame based analyte detector 14; and    -   means 16, 18 for introducing the analyte containing aerosol into        said flame based analyte detector 14;    -   in which the condensed phase produces no or negligible response        from flame based analyte detector 14; and        -   i) the system can operate at condensed phase flow rates of            greater than 20 μl min⁻¹, preferably greater than 50 μl            min⁻¹, most preferably greater than 100 μl min⁻¹;    -   and ii) the means 16, 18 for introducing the analyte containing        aerosol into said flame based detector 14 comprises a spray        chamber 16.

The system further comprises a box/oven (which can maintain atemperature controlled environment) 22 containing the detection system,which is securely mounted to increase stability. The condensed phaseflow enters a microconcentric nebuliser 12 through a narrow bore tube24, and the liquid is sprayed into a cyclonic spray chamber 16 with anebuliser gas, introduced via gas conducting conduit 26. The nebulisergas can be any suitable inert gas such as nitrogen or argon. Thecyclonic spray chamber 16 has a liquid drain 28 and connecting tubing30, which can be connected to a extraction peristaltic pump 32 orsimilar device to remove any condensed liquid. The aerosol spray thusformed exits the cyclonic chamber 16 at side arm outlet 16 a and passesinto the bottom end of a connecting tube 18. The connecting tube 18 canbe formed from any suitable material, such as steel or glass.Furthermore, the connection between the spray chamber 16 and connectingtube 18 does not have to be permanent, and in fact the system may bedemountable. The connecting tube 18 passes through a heated block 36 andintroduces the aerosol to the jet 38 of the flame detector 14. Theheater block 36 can be controlled up to a temperature of 450° C.,although the exact limit is not critical and may be largely in line withcurrent FID designs. In fact, block temperatures in excess ofconventionally used temperatures might be utilised in conjunction withthe present invention. Hydrogen is added to the aerosol flow at asimilar point to that of a conventional FID, but a higher flow rate ofhydrogen than is usual is required if the condensed phase is aqueous,because the aqueous aerosol flow cools the flame. This necessitates arelatively high hydrogen flow, and the use of wider than normal mixingslots. A hydrogen flow rate of ca. 100 ml min⁻¹ has been found to besuitable, although it is unlikely that this value is a critical one. Thejet of the flame based detector 14 comprises a wide bore alumina tube38. It is also possible to use a different ceramic or another material.The internal diameter of the alumina tube 38 is ca. 1.5 mm, although awider range of internal diameters, perhaps 1 to 3 mm, could be useddepending on the gas flow employed.

Air is added around the flame as in conventional FID systems in order tomaintain combustion. Flow rates are generally higher than inconventional FIDs; a representative value is 500 ml min⁻¹. A detectionsignal is generated by maintaining a potential difference of ca. 170Vbetween the jet and a collector (not shown).

In a representative, but non-limiting, example the analyte is dissolvedin an aqueous solution which is introduced into the nebuliser at a flowrate of between 0.01 and 1.0 ml min⁻¹ through a capillary tube. Higherflow rates, up to ca 3.0 ml min⁻¹, might be contemplated. A typical flowrate of nebuliser gas is ca. 400 ml min⁻¹. The aqueous solution cancontain limited quantities of dissolved inorganic salts, acids or basesto control pH and perform other known functions, provided that theseadditives do not result in any significant interfering signal from theFID.

The spray chamber 16 is an expansion chamber which is shown in moredetail in FIG. 2, in which like numerals are used to denote featureswhich are identical to features shown in FIG. 1. The spray chamber 16contains a centrifugal flow path from the periphery where the effluentflow enters to the central exit 16 b, coupled with a flow dimple 16 cwhich creates turbulence in the flow, thereby breaking up laminar flowso that larger droplets (which might cause spiking in the flame) hit thewalls and are lost, exiting via drain 28. Other ways of creatingturbulence would readily present themselves to the skilled person. Asecond flow dimple 16 d is disposed opposite the exit 16 b. Theconfiguration of the spray chamber is generally as described in Tayloret al, Journal of Analytical Atomic Spectrometry, 13 (1998) 1095-1100.

Water has been used in the separation stage as the condensed phase fromambient temperature up to ca. 250° C. When the separation temperature isgreater than 100° C. (superheated conditions), the condensed phase canbe produced by generating a back-pressure using a restriction in thetubing after the separation stage, so that the water is below itsboiling point when the water reaches the nebulisation means. Other waysof creating the back pressure include a frit or a mechanically operatedsolenoid. However, it should be noted that the condensed phase will notbe at such elevated temperatures when it reaches the nebulisation means.In fact, the condensed phase will be at a temperature substantiallybelow boiling point.

Furthermore, it should be noted that the temperature encountered by theeffluent at the nebuliser means is substantially less than the boilingpoint of the condensed phase. This overcomes a disadvantage ofthermospray type interfaces, namely that involatile or thermallyunstable analytes can be degraded or deposited and thus block the spraycapillary. It is possible to maintain the nebuliser means at a constant,but relatively low, temperature for stability purposes. In the case ofwater, the nebulisation means would be maintained at the temperature of50° C. or less.

The separation stage 10 is a LC system, although it is possible toutilise other separation stages such as capillary electrophoresis,electrochromatographic and capillary electrochromatographic stages.Other analyte analysis stages, such as flow injection analysis, might beused in place of a separation stage. Various LCs can be used inconjunction with the present invention: in particular, it is recognisedthat the internal diameters and internal volumes of the LC and theconduit which conveys the condensed phase to the nebuliser areadvantageously large enough to support flow rates in excess of 20,preferably greater then 50, most preferably greater than 100 μl min⁻¹.This is in contrast to the technique described in Hooijschuur et al.

In a specific embodiment, an LC system was constructed using a number ofmodular components. A Rheodyne 7161 injector was employed, and themobile phase was pumped using a Jasco PU980 pump. Columns from a rangeof manufacturers were used, having different internal diameters from 2mm to 4.6 mm, and lengths between 100 and 250 mm. The columns werepacked with a number of different stationary phases. The columns wereheated in either a Jones Chromatography Column oven (for thetemperatures up to 100° C.) or a Pye Unicam 104 GC oven for highertemperatures (or if temperature gradients are employed). Optionally, aHPLC type UV spectroscopic detector could be placed between the columnand the FID. A CETAC MCN-100-microconcentric nebuliser was employed(CETAC Technologies, Omaha, USA), although other nebulisers might beadvantageously employed instead.

FIGS. 3 to 5 show chromatograms obtained in a number of experiments,using the apparatus of the specific embodiment. FIG. 3 shows theseparation of the carbohydrate maltose, glucose and arabinose usingaqueous solution at 35° C. and a flow rate of 0.5 ml min⁻¹. FIG. 4 showsthe separation of the amino acids serine, arginine, proline, valine,methionine and isoleucine using aqueous solution (with 0.02%trifluoroacetic acid) at ambient temperature and a flow rate of 0.5 mlmin⁻¹. It is not possible to achieve these carbohydrate and amino acidseparations using gas chromatography owing to the involatility of thespecies. HPLC is not easy to perform because these species lackchromophores which are required for spectroscopic detection. FIG. 5shows the separation of benzaldehyde using superheated water at 200° C.and a flow rate of 0.2 ml min⁻¹.

In comparison to the nebuliser “comparison” experiment of Hooijschuur etal, the present system has the advantage of being able to handle muchhigher liquid flow rates, thereby increasing the versatility andsensitivity of the system. Furthermore, a higher nebuliser gas flow isused. This has the consequence that the gas flow itself, rather thangravity, is used to transport the aerosol droplets to the detectorflame, thereby permitting the detector to be mounted in the conventionalmanner, above the aerosol introduction point, rather than in theinverted configuration of Hooijschuur et al. Furtherstill, the provisionof the spray chamber provides advantages in terms of draining excessliquid and removing larger aerosol droplets. However, it is possiblethat other nebulisation systems might be advantageously employed withinsystems of the present invention without necessitating the provision ofa spray chamber.

1. An analyte detection system comprising: an analyte analysis stagethrough which a condensed phase containing the analyte flows; nebulisermeans into which the output of the analyte analysis stage is introduced,the nebuliser means producing an analyte containing aerosol; a flamebased analyte detector; and means for introducing the analyte containingaerosol into said flame based analyte detector; and i) the sytems canoperate at condensed phase flow rates of greater than 20 μl min⁻¹,preferably greater than 50 μl min⁻¹, most preferably greater than 100 μlmin⁻¹; and/or ii) the means for introducing the analyte containingaerosol into said flame based detector comprises a spray chamber.
 2. Ananalyte detection system according to claim 1 in which the condensedphase is aqueous.
 3. An analyte detection system according to claim 2 inwhich the condensed phase is an aqueous solution.
 4. An analytedetection system according to claim 3 in which the condensed phase is asuperheated aqueous solution.
 5. An analyte detection system accordingto claim 1 in which the condensed phase is a supercritical fluid.
 6. Ananalyte detection system according to claim 1 in which the condensedphase is a superheated liquid phase.
 7. An analyte detection systemaccording to claim 1 in which the detector is a flame ionisationdetector.
 8. An analyte detection system according to claim 1 in whichthe detector is a thermionic detector, pulsed flame photometric detectoror flame photometric detector.
 9. An analyte detection system accordingto claim 1 in which the analyte analysis stage comprises an analyteseparation state and effluent from the analyte separation stage isintroduced into the nebulisation means.
 10. An analyte detection systemaccording to claim 9 in which the analyte separation stage comprises aliquid chromatographic stage.
 11. An analyte detection system accordingto claim 9 in which the separation stage comprises a capillaryelectrophoresis, electrochromatographic or capillaryelectrochromatographic stage.
 12. An analyte detection system accordingto claim 1 in which the analyte analysis stage comprises a flowinjection analysis stage.
 13. An analyte detection system according toclaim 1 in which the system comprises a spray chamber in whichseparation of the aerosol is achieved so that larger aerosol dropletsare not introduced to the flame based analyte detector.
 14. An analytedetection system according to claim 13 in which the spray chamber isadapted to create turbulence in the flow of the aerosol.
 15. An analytedetection sytem according to claim 13 or claim 14 in which the spraychamber is adapted so as to cause the aerosol to flow in a centrifugalflow pattern.
 16. Use of a system according to claim 1 to detectvolatile and non-volatile compounds or elements.
 17. Use of a systemaccording to claim 1 to detect organic compounds.
 18. Use of a systemaccording to claim 1 to detect inorganic compounds.